Compositions and methods of use of phorbol esters in the treatment of neoplasms

ABSTRACT

Methods and compositions containing a phorbol ester or a derivative of a phorbol ester are provided for the treatment of chronic and acute conditions. Such conditions may be caused by disease, be symptoms, treatments, or sequelae of disease. The phorbol esters described are particularly useful in the treatment of neoplastic diseases and/or managing the side effects of chemotherapeutic and radiotherapeutic treatments of neoplastic diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/938,182, filed Mar. 28, 2018 (now allowed), which claims priority asa CONTINUATION of U.S. patent application Ser. No. 14/026,473 (now U.S.Pat. No. 9,974,764), which is a CONTINUATION-IN-PART OF U.S. applicationSer. No. 13/745,745, filed Jan. 18, 2013 (abandoned), which claimsbenefit of U.S. Provisional Patent Application Ser. No. 61/588,165,filed Jan. 18, 2012; and is a CONTINUATION-IN-PART OF U.S. applicationSer. No. 13/794,467, filed Mar. 11, 2013 (now U.S. Pat. No. 9,132,113),which is a CONTINUATION of U.S. patent application Ser. No. 13/595,072,filed Aug. 27, 2012 (abandoned), which is a CONTINUATION of U.S. patentapplication Ser. No. 12/023,753, filed Jan. 31, 2008 (abandoned), whichclaims benefit of U.S. Provisional Patent Application Ser. No.60/898,810, filed Jan. 31, 2007; the disclosures of which areincorporated herein in their entirety by reference.

ADDITIONAL DISCLOSURES

Additional disclosures relating to the instant application may be foundin “Compositions And Methods Of Use Of Phorbol Esters” U.S. patentapplication Ser. No. 12/023,753, filed Jan. 31, 2008, to Richard L.Chang, et al, which claims priority benefit of U.S. Provisional PatentApplication Ser. No. 60/898,810, filed Jan. 31, 2007, each of which isincorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

The present invention relates generally to the use of phorbol esters.Specifically, the present invention relates to the use of phorbol estersin the treatment and prevention of neoplasms and the management of sideeffects from radiation and chemotherapeutic treatment of neoplasms.

BACKGROUND

Plants have historically served many medicinal purposes. The WorldHealth Organization (WHO) estimates that 4 billion people, 80% of theworld population, presently use herbal medicine for some aspect ofprimary health care. (WHO Fact sheet Fact sheet No 134 December 2008)However, it can be difficult to isolate the specific compound that hasthe medicinal effect and to reproduce it on a commercial scale.Additionally, while active compounds may be isolated from a plant, theother parts of a plant such as the minerals, vitamins, volatile oils,glycosides, alkaloids, bioflavanoids, and other substances may also beinvolved in the functioning of the active ingredient or the medicinaleffect for which the plant is known, making the use, purification andcommercialization of plant based pharmaceutical agents a challenge.

Phorbol is a natural, plant-derived organic compound of the tiglianefamily of diterpenes. It was first isolated in 1934 as a hydrolysisproduct of croton oil derived from the seeds of Crown tiglium, a leafyshrub of the Euphorbiaceae family that is native to Southeastern Asia.Various esters of phorbol have important biological properties includingthe reported ability to mimic diacylglycerols and activate proteinkinase C (PKC); and to modulate downstream cell signaling pathwaysincluding the mitogen-activated protein kinase (MAPK) pathways. Phorbolesters are additionally thought to bind to chimaerins, the Ras activatorRasGRP, and the vesicle-priming protein Munc-13 (Brose N, Rosenmund C.,J Cell Sci; 115:4399-411 (2002)). Some phorbol esters also inducenuclear factor-kappa B (NF-κB). The most notable physiological propertyof phorbol esters is their reported capacity to act as tumor promoters.(Blumberg, 1988; Goel, G et al., Int, Journal of Toxicology 26, 279-288(2007)).

12-O-tetradecanoylphorbol-13-acetate (TPA), also calledphorbol-12-myristate-13-acetate (PMA), is a phorbol ester used in modelsof carcinogenesis as an inducer for differentiation and/or apoptosis inmultiple cell lines and primary cells. TPA has also been reported tocause an increase in circulating white blood cells and neutrophils inpatients whose bone marrow function has been depressed by chemotherapy(Han Z. T. et al. Proc. Natl. Acad. Sci. 95, 5363-5365 (1998)). However,due to a variety of factors, including caustic reactions when contactedwith the skin and concerns for its potential toxicity, TPA has not beenshown to be an effective adjuvant to chemotherapy. Indeed, as phorbolesters play a key role in activation of protein kinase C, which triggersvarious cellular responses resulting in inflammatory responses and tumordevelopment (Goel et al., Int, Journal of Toxicology 26, 279-288(2007)), phorbol esters would generally be excluded from possibletreatment candidates for neoplasms including cancer.

Cancer is one of the leading causes of death worldwide accounting for7.6 million deaths (around 13% of all deaths) in 2008 (GLOBOCAN 2008(IARC) Section of Cancer Information (Aug. 12, 2011)). Globally,12,662,600 new cases were diagnosed in 2008. (2008 (GLOBOCAN 2008 (IARC)Section of Cancer Information (Aug. 12, 2011)). In the U.S. alone,1,596,670 new cases of cancer were diagnosed in 2011 (Cancer Facts &Figures—2011, American Cancer Society (ACS), Atlanta, Ga., 2011).

Cancer treatments generally involve a combination of surgery,chemotherapy, hormonal therapy and/or radiation treatment to eradicateneoplastic cells in a patient. However, current therapeutics forneoplasms have a number of drawbacks including insufficient potency andintolerable side effects. Surgery, for example, may be contraindicateddue to the health of a patient. Additionally, it may be difficult toobtain clear margins around a tumor, resulting in some neoplastic tissuebeing left behind and an increased chance of recurrence of the disease.

Generally, chemotherapeutics act by killing cells that divide rapidly,one of the main properties of most cancer cells. However, they also harmnormal cells that divide rapidly such as cells in bone marrow, thedigestive tract and hair follicles. They frequently have significantside effects including severe nausea, bone marrow depression, andimmunosuppression.

Ionizing radiation works by damaging the DNA of exposed tissue. However,while targeted, it can still damage normal cells as well as neoplasmsand can have side effects such as anemia, nausea and vomiting, poorappetite, weight loss, constipation, diarrhea, hair loss, andinfertility.

For many patients, the toxic side effects of current therapies diminishtheir quality of life to such an extent they simply stop taking theirmedications. For others, therapeutic schedules are so complicated andinconvenient that compliance is limited. Other patients experienceexcellent results initially, but suffer relapses despite full compliancewith therapeutic regimens. There is clearly a need for new and moreeffective treatments for neoplasms and to manage the side effects ofcurrent treatments for neoplasms including cancer.

SUMMARY

The present invention relates to compositions containing and methods ofusing phorbol esters. The compositions and methods described herein areeffective in treating neoplastic conditions and in managing side effectsfrom chemotherapeutic or radiation treatment of neoplastic conditions.Such neoplasms may be malignant or benign. In some embodiments,neoplasms may be solid or non-solid cancers. In other embodiments, theneoplasms may be relapses. In another embodiment, the neoplasms may berefractory.

Exemplary neoplasms include, but are not limited to, hematologicmalignancies/bone marrow disorders, including, but not limited to,leukemia, including acute myeloid leukemia (AML), chronic myeloidleukemia (CML), chronic myeloid leukemia blast crisis, myelodysplasia,and myeloproliferative syndrome; lymphoma, including Hodgkin's andnon-Hodgkin's lymphoma; subcutaneous adenocarcinoma; ovarianteratocarcinoma; liver cancer; breast cancer; bone cancer; lung cancer;pancreatic cancer; non-small cell lung cancer; and prostate cancer.Other neoplastic conditions amenable to treatment using the methods andcompositions as described herein include other cancer disorders andconditions, including solid tumors of various types.

Compositions and methods herein may additionally be used treat symptomsof neoplastic disease including, but not limited to, anemia; chronicfatigue; excessive or easy bleeding, such as bleeding of the nose, gums,and under the skin; easy bruising, particularly bruising with noapparent cause; shortness of breath; petechiae; recurrent fever; swollengums; slow healing of cuts; bone and joint discomfort; recurrentinfections; weight loss; itching; night sweats; lymph node swelling;fever; abdominal pain and discomfort; disturbances in vision; coughing;loss of appetite; pain in the chest; difficulty swallowing; swelling ofthe face, neck and upper extremities; a need to urinate frequently,especially at night; difficulty starting urination or holding backurine; weak or interrupted flow of urine; painful or burning urination;difficulty in having an erection; painful ejaculation; blood in urine orsemen; frequent pain or stiffness in the lower back, hips, or upperthighs; and weakness.

Successful treatment and/or remission will be determined according toconventional methods, such as determining size reduction of solidtumors, and/or histopathological studies to assess growth, stage,metastatic state or potential, presence or expression levels ofhistological cancer markers, decrease in symptoms etc.

Compositions and methods herein may further be used to treat or preventthe side effects of chemotherapy and radiation therapy which arecommonly used as treatments for neoplastic disease. Such side effectsinclude, but not limited to, alopecia, nausea, vomiting, poor appetite,soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue,constipation, oral ulcers, itchy skin, peeling, nerve and muscle damage,auditory changes, weight loss, diarrhea, immunosuppression, bruising,heart damage, bleeding, liver damage, kidney damage, edema, mouth andthroat sores, infertility, fibrosis, epilation, moist desquamation,mucosal dryness, vertigo and encephalopathy.

In yet another embodiment, the phorbol esters and derivatives of phorbolesters as described herein may be used to modulate cell signalingpathways. Such modulation may have a variety of results, for example, insome embodiments, the use of compositions containing phorbol esters andderivatives of phorbol esters may increase white blood cell counts inmammalian subjects. In another embodiment, compositions containingphorbol esters and/or phorbol ester derivatives may alter the release ofTh1 cytokines in mammalian subjects. In a further embodiment,compositions containing phorbol esters and/or phorbol ester derivativesmay alter the release of interleukin 2 (IL-2) in mammalian subjects. Inan additional embodiment, compositions containing phorbol esters and/orphorbol ester derivatives may alter the release of interferon inmammalian subjects. In yet another embodiment, compositions containingphorbol esters and/or phorbol ester derivatives may alter the rate ofERK phosphorylation.

The invention achieves the foregoing and satisfies additional objectsand advantages by providing novel and surprisingly effective methods andcompositions for modulating cell signaling pathways and/or treatingdiseases, symptoms of diseases or managing side effects from treatmentsof diseases using compositions containing a phorbol ester of Formula I,below:

wherein R₁ and R₂ may be hydrogen; hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof. R₃ may be hydrogen or

and substituted derivatives thereof. The methods and compositions of thepresent invention further include any pharmaceutical salts, enantiomers,isomer, polymorphs, prodrugs, hydrates and solvates of the compositionsof Formula I.

In some embodiments, at least one of R₁ and R₂ are other than hydrogenand R₃ is hydrogen or

and substituted derivatives thereof. In another embodiment, either R₁ orR₂ is

the remaining R₁ or R₂ is a

wherein a lower alkyl is between 1 and 7 carbons, and R₃ is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino; and/or similar type radicals.

The invention achieves these objects and satisfies additional objectsand advantages by providing novel and surprisingly effective methods andcompositions for modulating cell signaling pathways and/or treatingneoplasms or side effects from chemotherapeutic or radiotherapytreatments of neoplasms using an exemplary phorbol ester compositionsuch as 12-O-tetradecanoylphorbol-13-acetate (TPA) of Formula II, below:

Useful phorbol esters of Formula I and related compounds and derivativeswithin the formulations and methods of the invention include, but arenot limited to, other pharmaceutically acceptable active salts of saidcompounds, as well as active isomers, enantiomers, polymorphs,glycosylated derivatives, solvates, hydrates, and/or prodrugs of saidcompounds. Exemplary forms of phorbol esters for use within thecompositions and methods of the invention include, but are not limitedto, phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate;phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate;phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol12-myristate; phorbol 13-myristate; phorbol 12-myristate-13-acetate(also known as TPA or PMA); phorbol 12,13,20-triacetate; 12-deoxyphorbol13-angelate; 12-deoxyphorbol 13-angelate 20-acetate; 12-deoxyphorbol13-isobutyrate; 12-deoxyphorbol 13-isobutyrate-20-acetate;12-deoxyphorbol 13-phenylacetate; 12-deoxyphorbol 13-phenylacetate20-acetate; 12-deoxyphorbol 13-tetradecanoate; phorbol 12-tigliate13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; andphorbol 13-acetate.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI or derivative of a phorbol ester of the Formula I, particularly TPA,according to the methods of the invention include, but are not limitedto, subjects suffering from neoplastic diseases including malignantneoplastic diseases such as solid and non-solid cancers. Non-solidcancers may include, hematologic malignancies/bone marrow disorders,including, but not limited to, leukemia, including acute myeloidleukemia (AML), chronic myeloid leukemia (CML), chronic myeloid leukemiablast crisis, myelodysplasia, myeloproliferative syndrome. Solid cancersmay include, but are not limited to, lymphoma, including Hodgkin's andnon-Hodgkin's lymphoma, subcutaneous adenocarcinoma, ovarianteratocarcinoma, lung cancer; bone cancer; breast cancer; liver cancer;pancreatic cancer; oral cancer; non-small cell lung cancer and prostatecancer.

Subjects amenable to treatment with phorbol esters of Formula I,particularly TPA, or derivatives of the phorbol esters of the Formula Iincluding pharmaceutically acceptable salts, enantiomers, isomer,polymorphs, prodgrugs, solvates and hydrates further include thosesuffering from symptoms of such neoplastic diseases such as, but notlimited to, anemia; chronic fatigue; excessive or easy bleeding, such asbleeding of the nose, gums, and under the skin; easy bruising,particularly bruising with no apparent cause; shortness of breath;petechiae; recurrent fever; swollen gums; slow healing of cuts; bone andjoint discomfort; recurrent infections; weight loss; itching; nightsweats; lymph node swelling; fever; abdominal pain and discomfort;disturbances in vision; coughing; loss of appetite; pain in the chest;difficulty swallowing; swelling of the face, neck and upper extremities;a need to urinate frequently, especially at night; difficulty startingurination or holding back urine; weak or interrupted flow of urine;painful or burning urination; difficulty in having an erection; painfulejaculation; blood in urine or semen; frequent pain or stiffness in thelower back, hips, or upper thighs; and weakness. In some embodiments,such cancers may be relapses or refractory.

Further mammalian subjects that are amenable to treatment with phorbolesters of Formula I, or derivative of the phorbol esters of the FormulaI, particularly TPA, according to the methods of the present inventioninclude, but are not limited to, subjects suffering from side effects ofchemotherapy or radiation therapy for the treatment of neoplasticdiseases including malignant neoplastic diseases such as solid andnon-solid cancers. Such side effects include, but are not limited to,alopecia, nausea, vomiting, poor appetite, soreness, neutropenia,anemia, thrombocytopenia, dizziness, fatigue, constipation, oral ulcers,itchy skin, peeling, nerve and muscle damage, auditory changes, weightloss, diarrhea, immunosuppression, bruising, heart damage, bleeding,liver damage, kidney damage, edema, mouth and throat sores, infertility,fibrosis, epilation, and moist desquamation, mucosal dryness, vertigoand encephalopathy.

These and other subjects are effectively treated prophylactically and/ortherapeutically, by administering to the subject an effective amount ofa phorbol ester of Formula I or derivative of a phorbol ester of FormulaI sufficient to decrease and/or eliminate neoplastic cells, increasewhite blood cell counts, induce remission, maintain remission, preventor reduce symptoms and conditions associated with malignancies, increaseERK phosphorylation, modulate NF-κB activity, increase Th1 cytokineactivity, decrease or eliminate radiation damage, boost the immunesystem, decrease nausea, decrease or prevent hair loss, increaseappetite, decrease soreness, increase energy levels, relievegastrointestinal distress, decrease bruising, eliminate oral ulcers,decrease or eliminate skin damage due to radiation, increase or maintainneutrophil levels, increase or maintain platelet levels, decrease edema,and/or decrease or eliminate moist desquamation.

Therapeutically useful methods and formulations of the invention willeffectively use a phorbol ester of Formula I in a variety of forms, asnoted above, including any active, pharmaceutically acceptable salts ofsaid compounds, as well as active isomers, enantiomers, polymorphs,solvates, hydrates, prodrugs, and/or combinations thereof. TPA offormula II is employed as an illustrative embodiment of the inventionwithin the examples herein below.

Within additional aspects of the invention, combinatorial formulationsand methods are provided which employ an effective amount of a phorbolester of Formula I or derivative of a phorbol ester of Formula I incombination with one or more secondary or adjunctive active agent(s)that is/are combinatorially formulated or coordinately administered withthe phorbol ester compound of Formula I to yield an effective responsein the subject.

Exemplary combinatorial formulations and coordinate treatment methods inthe treatment of neoplastic disease employ a phorbol ester compound ofFormula I or derivative of the phorbol ester of Formula I in combinationwith one or more additional, neoplastic disease treating or otherindicated, secondary or adjunctive therapeutic agents. The secondary oradjunctive therapeutic agents used in combination with a phorbol ester,e.g., TPA, in these embodiments may possess direct or indirectchemotherapeutic effects, alone or in combination with, e.g. TPA; mayexhibit other useful adjunctive therapeutic activity in combination witha phorbol ester, e.g. TPA (such as cytotoxic, anti-inflammatory, NF-κBinhibiting, apoptosis inducing, Th1 cytokine increasing activity); ormay exhibit adjunctive therapeutic activity useful for treatingneoplasms or associated symptoms alone or in combination with, e.g. TPA.

Useful adjunctive or secondary therapeutic agents in these combinatorialformulations and coordinate treatment methods for the treatment ofneoplastic diseases include doxorubicin, vitamin D3, cytarabine,cytosine arabinoside, daunorubicin, cyclophosphamide, gemtuzumabozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine,aldesleukin, asparaginase, carboplatin, etoposide phosphate,fludarabine, methotrexate, etoposide, dexamethasone, and cholinemagnesium trisalicylate. In addition, adjunctive or secondary therapiesmay be used such as, but not limited to, radiation treatment, hormonetherapy and surgery.

Exemplary combinatorial formulations and coordinate treatment methods inthe prevention or treatment of side effects from chemotherapy employ aphorbol ester compound of Formula I or derivative of the phorbol esterof Formula I in combination with one or more additional, chemoprotectiveor other indicated, secondary or adjunctive therapeutic agents. Thesecondary or adjunctive therapeutic agents used in combination with thephorbol ester, e.g., TPA, in these embodiments may possess direct orindirect chemoprotective effects, alone or in combination with thephorbol ester, e.g. TPA; may exhibit other useful adjunctive therapeuticactivity in combination with a phorbol ester, e.g. TPA (such asanti-inflammatory, neutrophil stimulating, erythropoiesis stimulating,bone resorption inhibiting, bone strengthening, antiemetic, painrelieving); or may exhibit adjunctive therapeutic activity useful fortreating or preventing side effects of chemotherapy or associatedsymptoms alone or in combination with a phorbol ester, e.g. TPA.

Useful adjunctive or secondary therapeutic agents in these combinatorialformulations and coordinate treatment methods for the prevention ortreatment of side effects of chemotherapy in a mammalian subjectinclude, but are not limited to, pegfilgrastim, epoeitn alfa,darbepoetin alfa, alendronate sodium, risedronate, ibandronate, G-CSF,5-HT₃ receptor antagonists, NK₁ antagonists, olanzapine,corticosteroids, dopamine antagonists, serotonin antagonists,benzodiazepines, aprepitant, and cannabinoids.

Exemplary combinatorial formulations and coordinate treatment methods inthe prevention or treatment of side effects from radiation therapy ascontemplated herein employ a phorbol ester compound of Formula I orderivative of the phorbol ester of Formula I in combination with one ormore additional, radiation protective or other indicated, secondary oradjunctive therapeutic agents. The secondary or adjunctive therapeuticagents used in combination with a phorbol ester e.g., TPA, in theseembodiments may possess direct or indirect protection from radiationdamage, alone or in combination with a phorbol ester, e.g. TPA; mayexhibit other useful adjunctive therapeutic activity in combination withthe phorbol ester, e.g. TPA (such as anti-swelling, cytoprotective,anti-mucositis, epithelial stimulating, anti-fibrotic, plateletstimulating); or may exhibit adjunctive therapeutic activity useful fortreating or preventing side effects of radiation therapy or associatedsymptoms alone or in combination with, e.g. TPA.

Useful adjunctive or secondary therapeutic agents in these combinatorialformulations and coordinate treatment methods for the prevention ortreatment of side effects of radiation therapy in a mammalian subjectinclude, but are not limited to, steroids, amifostine, chlorhexidine,benzydamine, sucralfate, keratinocyte growth factor (KGF), palifermin,Cu/Zn superoxide dismutase, Interleukin 11, or prostaglandins.

The forgoing and additional objects, features, aspects and advantages ofthe present invention will become apparent from the following detaileddescription.

DETAILED DESCRIPTION

Novel methods and compositions have been identified for use inpreventing and/or treating neoplastic diseases and conditions inmammalian subjects. In various embodiments, the methods and compositionsare effective to prevent or treat neoplastic diseases and symptoms ofsuch diseases as well as side effects from chemotherapeutic andradiotherapeutic treatments of such diseases. Such neoplastic diseasesmay or may not be malignant. In some embodiments, the neoplasticdiseases may be solid or non-solid cancers. In other embodiments, thecancers may be refractory or relapses.

In additional embodiments, the methods and compositions are effective inpreventing or ameliorating damage or side effects from chemotherapeuticagents. In further embodiments, the methods and compositions asdescribed herein are effective in preventing or ameliorating damage orside effects from radiation therapy. The composition and methods asdescribed herein may increase immune responsiveness, increase therelease of Th1 cytokines, decrease and/or eliminate neoplastic cells,increase white blood cell counts, induce remission, maintain remission,prevent or reduce symptoms and conditions associated with malignancies,increase ERK phosphorylation, modulate NF-κB activity, decrease oreliminate radiation damage, boost the immune system, decrease nausea,decrease or prevent hair loss, increase appetite, decrease soreness,increase energy levels, relieve gastrointestinal distress, decreasebruising, eliminate oral ulcers, decrease or eliminate skin damage dueto radiation, increase or maintain neutrophil levels, increase ormaintain platelet levels, decrease edema, and/or decrease or eliminatemoist desquamation.

Formulations and methods provided herein employ a phorbol ester ofFormula I or derivative compound of a phorbol ester of Formula I as morefully described in U.S. patent application Ser. No. 12/023,753, filedJan. 31, 2008, which claims priority benefit of U.S. Provisional patentapplication Ser. No. 60/898,810, filed Jan. 31, 2007, each of which isincorporated herein in its entirety by reference,

wherein R₁ and R₂ may be hydrogen; hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof. R₃ may be hydrogen or

In some embodiments, at least one of R₁ and R₂ are other than hydrogenand R₃ is hydrogen or

and substituted derivatives thereof. In another embodiment, either R₁ orR₂ is

the remaining R₁ or R₂ is a

wherein a lower alkyl is between 1 and 7 carbons, and R₃ is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino; and/or similar type radicals.

Such compositions and methods additionally include all activepharmaceutically acceptable compounds of this description as well asvarious foreseen and readily provided complexes, salts, solvates,isomers, enantiomers, polymorphs and prodrugs of the compounds ofFormula I and combinations thereof as neoplastic disease treatingcompounds and/or as treatments for the management of side effects fromchemotherapeutic or radiotherapeutic treatments for neoplasms.

Immune responsiveness increasing formulations and methods providedherein employ a phorbol ester of Formula I or related salt, solvate,isomer, enantiomer, polymorph or prodrug of a compound of Formula I, andcombinations thereof as immune stimulatory compounds.

Th1 cytokine increasing formulations and methods provided herein employa phorbol ester or derivative compound of Formula I, above, includingall active pharmaceutically acceptable compounds of this description aswell as various foreseen and readily provided complexes, salts,solvates, isomers, enantiomers, polymorphs and prodrugs of thesecompounds and combinations thereof as novel Th1 cytokine increasingagents.

Formulations and methods provided herein additionally employ a phorbolester or derivative compound of Formula I, above, including all activepharmaceutically acceptable compounds of this description as well asvarious foreseen and readily provided complexes, salts, solvates,isomers, enantiomers, polymorphs and prodrugs of these compounds andcombinations thereof in the treatment of neoplastic diseases.

Apoptosis inducing formulations and methods provided herein employ aphorbol of Formula I or derivative compound of a phorbol ester ofFormula I, above, including all active pharmaceutically acceptablecompounds of this description as well as various foreseen and readilyprovided complexes, salts, solvates, isomers, enantiomers, polymorphsand prodrugs of these compounds and combinations thereof aschemotherapeutic agents that induce apoptosis in neoplasms.

Remission inducing formulations and methods provided herein employ aphorbol of Formula I or derivative compound of a phorbol ester ofFormula I, above, including all active pharmaceutically acceptablecompounds of this description as well as various foreseen and readilyprovided complexes, salts, solvates, isomers, enantiomers, polymorphsand prodrugs of these compounds and combinations thereof asanti-neoplasm agents.

Formulations and methods provided herein further employ a phorbol esterof Formula I or derivative compound of a phorbol ester of Formula I,above, including all active pharmaceutically acceptable compounds ofthis description as well as various foreseen and readily providedcomplexes, salts, solvates, isomers, enantiomers, polymorphs andprodrugs of these compounds and combinations thereof in the preventionor treatment of side effects from chemotherapy.

Formulations and methods provided herein additionally employ a phorbolester of Formula I or derivative compound of a phorbol ester of FormulaI, above, including all active pharmaceutically acceptable compounds ofthis description as well as various foreseen and readily providedcomplexes, salts, solvates, isomers, enantiomers, polymorphs andprodrugs of these compounds and combinations thereof in the preventionor treatment of side effects from radiation therapy.

A broad range of mammalian subjects, including human subjects, areamenable to treatment using the formulations and methods of theinvention. These subjects include, but are not limited to, individualssuffering from diseases or conditions including neoplastic diseases orside effects from chemotherapeutic or radiotherapeutic treatment ofneoplastic diseases.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI or derivative compounds of a phorbol ester of Formula I, particularlyTPA, according to the methods of the present invention additionallyinclude, but are not limited to, mammalian subjects with neoplasticdiseases including solid and non-solid cancers, including hematologicmalignancies/bone marrow disorders, such as leukemia, including acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myeloidleukemia blast crisis, myelodysplasia, myeloproliferative syndrome;lymphoma, including Hodgkin's and non-Hodgkin's lymphoma; subcutaneousadenocarcinoma; ovarian teratocarcinoma; breast cancer; lung cancer;liver cancer; and prostate cancer. In some embodiments, such cancers maybe relapses or refractory.

Within the methods and compositions of the invention, one or morephorbol ester compound(s) of Formula I or derivative compounds of aphorbol ester of Formula I as disclosed herein is/are effectivelyformulated or administered as an agent effective for treating neoplasticdiseases. In exemplary embodiments, TPA is demonstrated for illustrativepurposes to be an effective agent in pharmaceutical formulations andtherapeutic methods, alone or in combination with one or more adjunctivetherapeutic agent(s). The present disclosure further providesadditional, pharmaceutically acceptable phorbol ester compounds in theform of a native or synthetic compound, including complexes,derivatives, salts, solvates, isomers, enantiomers, polymorphs, andprodrugs of the compounds disclosed herein, and combinations thereof,which are effective as therapeutic agents within the methods andcompositions of the invention in the treatment of neoplastic diseasesand symptoms of such diseases.

Neoplastic disease is any growth or tumor caused by abnormal anduncontrolled cell division; it may spread to other parts of the bodythrough the lymphatic system or the blood stream. Such growths may bemalignant or benign, solid or non-solid.

In some embodiments, the neoplastic diseases amenable to treatment orprevention by the compositions and methods as described herein may be ahematological neoplasm/bone marrow disorder such as acute myeloidleukemia (AML). AML (also called acute myelogenous leukemia, acutemyeloblastic leukemia, acute granulocytic leukemia, and acutenonlymphocytic leukemia) is the most common type of acute leukemia inadults. In AML, stem cells produced by the bone marrow usually developinto a type of immature white blood cell called myeloblasts (or myeloidblasts). In individuals suffering from AML, these myeloblasts do notmature into healthy white blood cells. Additionally, stem cells inindividuals with AML may develop into abnormal red blood cells orplatelets. The lack of normal blood cells increases incidences ofinfection, anemia, and easy bleeding. Additionally, the leukemia cellscan spread outside the blood to other parts of the body, including thecentral nervous system (brain and spinal cord), skin, and gums.

The average age of a patient with AML is over 64 years of age. Remissionrates are inversely related to age with patients over the age of 70treated for AML with standard chemotherapeutics having a remission rateof less than 26%. Additionally, patients who develop AML after anantecedent hematologic disorder or prior leukemogenicchemotherapy/radiation therapy have similarly poor outcomes.

In other embodiments, the neoplasm treated by the compositions andmethods as described herein may include, but are not limited to, breastcancer, lung cancer, liver cancer, bone cancer, pancreatic cancer, nonsmall cell lung cancer, oral cancer as well as forms of lymphoma.

Chemotherapy is the treatment of cancer with an anti-neoplastic drug orcombination of such drugs. Chemotherapy works by impairing thereproduction of rapidly splitting cells, a property common in cancerouscells. However it does not actively distinguish between healthy cellsthat are also rapidly splitting and cancerous cells and it has a numberof side effects such as, but not limited to, alopecia, nausea, vomiting,poor appetite, soreness, neutropenia, anemia, thrombocytopenia,dizziness, fatigue, constipation, oral ulcers, itchy skin, peeling,nerve and muscle leprosy, auditory changes, problems with blood, weightloss, diarrhea, immunosuppression, bruising, tendency to bleed easily,heart damage, liver damage, kidney damage, vertigo and encephalopathy.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI, particularly TPA, according to the methods of the present inventionadditionally include, but are not limited to, mammalian subjectsundergoing chemotherapy.

Within the methods and compositions of the invention, one or morephorbol ester compound(s) of Formula I or derivative compounds of aphorbol ester of Formula I as disclosed herein is/are effectivelyformulated or administered as an agent effective for preventing ortreating side effects due to chemotherapy. In exemplary embodiments, TPAis demonstrated for illustrative purposes to be an effective agent inpharmaceutical formulations and therapeutic methods, alone or incombination with one or more adjunctive therapeutic agent(s). Thepresent disclosure further provides additional, pharmaceuticallyacceptable phorbol ester compounds in the form of a native or syntheticcompound, including complexes, derivatives, salts, solvates, isomers,enantiomers, polymorphs, and prodrugs of the compounds disclosed herein,and combinations thereof, which are effective as therapeutic agentswithin the methods and compositions of the invention in the preventionor treatment of side effects due to chemotherapy.

Radiation therapy uses high-energy radiation to shrink tumors and killcancer cells. It may be applied externally, internally, or systemically.It can cause acute or chronic side effects. Acute side effects occurduring treatment, and chronic side effects occur months or even yearsafter treatment ends. The side effects that develop depend on the areaof the body being treated, the dose given per day, the total dose given,the patient's general medical condition, and other treatments given atthe same time. (National Cancer Institute, 2011). Common side effects ofradiation therapy are moist desquamation, soreness, diarrhea, nausea,vomiting, appetite loss, constipation, itchy skin, peeling, mouth andthroat sores, edema, infertility, fibrosis, epilation, and mucosaldryness.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI, particularly TPA, according to the methods of the present inventionadditionally include, but are not limited to, mammalian subjectsundergoing radiation therapy.

Within the methods and compositions of the invention, one or morephorbol ester compound(s) of Formula I or derivative compounds of aphorbol ester of Formula I as disclosed herein is/are effectivelyformulated or administered as an agent effective for preventing ortreating side effects due to radiation therapy. In exemplaryembodiments, TPA is demonstrated for illustrative purposes to be aneffective agent in pharmaceutical formulations and therapeutic methods,alone or in combination with one or more adjunctive therapeuticagent(s). The present disclosure further provides additional,pharmaceutically acceptable phorbol ester compounds in the form of anative or synthetic compound, including complexes, derivatives, salts,solvates, isomers, enantiomers, polymorphs, and prodrugs of thecompounds disclosed herein, and combinations thereof, which areeffective as therapeutic agents within the methods and compositions ofthe invention in the prevention or treatment of side effects due toradiation therapy.

Phorbol is a natural, plant-derived polycyclic alcohol of the tiglianefamily of diterpenes. It was first isolated in 1934 as the hydrolysisproduct of croton oil derived from the seeds of Crown tiglium. It iswell soluble in most polar organic solvents and in water. Esters ofphorbol have the general structure of Formula I, below:

wherein R₁ and R₂ are selected from the group consisting of hydrogen;hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof and R₃ may be hydrogen,

or substituted derivatives thereof as well as pharmaceuticallyacceptable salts, enantiomers, polymorphs, prodrugs solvates andhydrates of compounds of Formula I and substituted derivatives thereof.

The term “lower alkyl” or “lower alkenyl” as used herein means moietiescontaining 1 to 7 carbon atoms. In the compounds of the Formula I, thealkyl or alkenyl groups may be straight or branched chain. In someembodiments, either or both R₁ or R₂, are a long chain carbon moiety(i.e., Formula I is decanoate or myristate).

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino and similar type radicals.

Organic and synthetic forms of phorbol esters, including anypreparations or extracts from herbal sources such as crown tiglium, arecontemplated as useful compositions comprising phorbol esters (orphorbol ester analogs, related compounds and/or derivatives) for usewithin the embodiments herein. Useful phorbol esters and/or relatedcompounds for use within the embodiments herein will typically have astructure as illustrated in Formula I, although functionally equivalentanalogs, complexes, conjugates, and derivatives of such compounds willalso be appreciated by those skilled in the art as within the scope ofthe invention.

In more detailed embodiments, illustrative structural modificationsaccording to Formula I above will be selected to provide usefulcandidate compounds for treating and/or preventing neoplastic diseasesand/or managing or preventing side effects in individuals undergoingchemotherapeutic or radiotherapeutic treatments, wherein: at least oneof R₁ and R₂ are other than hydrogen and R₃ is selected from the groupconsisting of hydrogen or

and substituted derivatives thereof. In another embodiment, either R₁ orR₂ is

the remaining R₁ or R₂ is

and R₃ is hydrogen.

An exemplary embodiment of a phorbol ester compound of Formula I usefulin the treatment of neoplastic diseases, particularly AML, or themanagement of side effects from the treatment of neoplastic diseases isfound in phorbol 12-myristate-13-acetate (also known as PMA or12-O-tetradecanoyl-phorbol-13-acetate (TPA)) shown in Formula II, below.

Additional useful phorbol esters and related compounds and derivativeswithin the formulations and methods of the invention include, but arenot limited to, other pharmaceutically acceptable active salts of saidcompounds, as well as active isomers, enantiomers, polymorphs,glycosylated derivatives, solvates, hydrates, and/or prodrugs of saidcompounds. Derivatives of phorbol esters of Formula I may or may not bephorbol esters themselves. Further exemplary forms of phorbol esters foruse within the compositions and methods of the invention include, butare not limited to, phorbol 13-butyrate; phorbol 12-decanoate; phorbol13-decanoate; phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate;phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol12-myristate; phorbol 13-myristate; phorbol 12,13,20-triacetate;12-deoxyphorbol 13-angelate; 12-deoxyphorbol 13-angelate 20-acetate;12-deoxyphorbol 13-isobutyrate; 12-deoxyphorbol13-isobutyrate-20-acetate; 12-deoxyphorbol 13-phenylacetate;12-deoxyphorbol 13-phenylacetate 20-acetate; 12-deoxyphorbol13-tetradecanoate; phorbol 12-tigliate 13-decanoate; 12-deoxyphorbol13-acetate; phorbol 12-acetate; and phorbol 13-acetate as shown in Table1.

TABLE 1 Exemplary Phorbol Esters Phorbol 13-Butyrate

Phorbol 12-Decanoate

Phorbol 13-Decanoate

Phorbol 12,13- Diacetate

Phorbol 13,20- Diacetate

Phorbol 12,13- Dibenzoate

Phorbol 12,13- Dibutyrate

Phorbol 12,13- Didecanoate

Phorbol 12,13- Dihexanoate

Phorbol 12,13- Dipropionate

Phorbol 12-Myristate

Phorbol 13-Myristate

Phorbol 12-Myristate- 13-Acetate (also known as TPA or PMA)

Phorbol 12,13,20- Triacetate

12-Deoxyphorbol 13-Angelate

12-Deoxyphorbol 13-Angelate 20-Acetate

12-Deoxyphorbol 13-Isobutyrate

12-Deoxyphorbol 13-Isobutyrate- 20-Acetate

12-Deoxyphorbol 13-Phenylacetate

12-Deoxyphorbol 13-Phenylacetate 20-Acetate

12-Deoxyphorbol 13-Tetradecanoate

Phorbol 12-Tigliate 13-Decanoate

12-Deoxyphorbol 13-Acetate

Phorbol 12-Acetate

Phorbol 13-Acetate

Phorbol ester compositions and derivative compositions hereinadditionally may comprise chemotherapeutic compositions comprising ananti-neoplastic effective amount of a phorbol ester of Formula I orderivative compound of a phorbol ester of Formula I, which is effectivefor maintenance and treatment of malignancies or symptoms caused bycancer in a mammalian subject. A “chemotherapeutic”, “anti-tumor,”“cancer treating”, “apoptosis inducing”, “remission inducing”,“remission maintaining” effective amount of the active compound istherapeutically effective, in a single or multiple unit dosage form,over a specified period of therapeutic intervention, to measurablyalleviate one or more symptoms of malignancy in a subject, and/or toalleviate one or more symptom(s) or condition(s) associated withmalignancy in the subject. Within exemplary embodiments, thecompositions of the invention are effective in treatment methods toalleviate symptoms of neoplastic disease related conditions in human andother mammalian subjects vulnerable to malignancies.

Compositions as described herein comprise chemoprotective compositionscomprising an effective amount of a phorbol ester compound of Formula Ior derivative compound of a phorbol ester of Formula I includingpharmaceutically acceptable salts, enantiomers, isomers, polymorphs,prodrugs, hydrates and solvates thereof to prevent or alleviate the sideeffects of chemotherapy. A “chemoprotective,” “anti-inflammatory,”“neutrophil stimulating,” “erythropoiesis stimulating,” “bone resorbtioninhibiting,” “bone strengthening,” “antiemetic,” “pain relieving”effective amount of the active compound is therapeutically effective, ina single or multiple unit dosage form, over a specified period oftherapeutic intervention, to measurably alleviate one or more of theside effects of chemotherapy in a subject. Within exemplary embodiments,the compositions of the invention are effective in treatment methods toalleviate side effects of chemotherapy in human and other mammaliansubjects undergoing chemotherapy.

Compositions as described herein comprise radiation therapy protectivecompositions comprising an effective amount of a phorbol ester compoundof Formula I or derivative compound of phorbol esters of Formula Iincluding pharmaceutically acceptable salts, enantiomers, isomers,polymorphs, prodrugs, hydrates and solvates thereof to prevent oralleviate the side effects of radiation therapy. A “radiationprotective,” “radioprotective,” “anti-swelling,” “cytoprotective,”“anti-mucositis,” “epithelial stimulating,” “anti-fibrotic,” “plateletstimulating” effective amount of the active compound is therapeuticallyeffective, in single or multiple unit dosage form, over a specifiedperiod of therapeutic intervention, to measurably alleviate one or moreof the side effects of chemotherapy in a subject. Within exemplaryembodiments, the compositions of the invention are effective intreatment methods to alleviate side effects of radiation therapy inhuman and other mammalian subjects undergoing radiation therapy.

Phorbol ester treating, including chemotherapeutic, chemoprotectant,radioprotectant, Th1 cytokine increasing, ERK phosphorylation inducing,anti-tumor, cancer treating, remission inducing, remission maintaining,apoptosis inducing, NFκB modulating compositions of the inventiontypically comprise an effective amount or unit dosage of a phorbol estercompound of Formula I or derivative compound of a phorbol ester ofFormula I, which may be formulated with one or more pharmaceuticallyacceptable carriers, excipients, vehicles, emulsifiers, stabilizers,preservatives, buffers, and/or other additives that may enhancestability, delivery, absorption, half-life, efficacy, pharmacokinetics,and/or pharmacodynamics, reduce adverse side effects, or provide otheradvantages for pharmaceutical use. Effective amounts of a phorbol estercompound or related or derivative compound of Formula I (e.g., a unitdose comprising an effective concentration/amount of TPA, or of aselected pharmaceutically acceptable salt, isomer, enantiomer, solvate,polymorph and/or prodrug of TPA) will be readily determined by those ofordinary skill in the art, depending on clinical and patient-specificfactors. Suitable effective unit dosage amounts of the active compoundsfor administration to mammalian subjects, including humans, may rangefrom about 10 to about 1500 μg, about 20 to about 1000 μg, about 25 toabout 750 μg, about 50 to about 500 μg, about 150 to about 500 μg, about125 μg to about 500 μg, about 180 to about 500 μg, about 190 to about500 μg, about 220 to about 500 μg, about 240 to about 500 μg, about 260to about 500 μg, about 290 to about 500 μg. In certain embodiments, thedisease treating effective dosage of a phorbol ester compound or relatedor derivative compound of Formula I may be selected within narrowerranges of, for example, 10 to 25 μg, 30-50 μg, 75 to 100 μg, 100 to 300μg, or 150 to 500 μg. These and other effective unit dosage amounts maybe administered in a single dose, or in the form of multiple daily,weekly or monthly doses, for example in a dosing regimen comprising from1 to 5, or 2 to 3, doses administered per day, per week, or per month.In one exemplary embodiment, dosages of 10 to 30 μg, 30 to 50 μg, 50 to100 μg, 100 to 300 μg, or 300 to 500 μg, are administered one, two,three, four, or five times per day. In more detailed embodiments,dosages of 50-100 μg, 100-300 μg, 300-400 μg, or 400-600 μg areadministered once or twice daily. In a further embodiment, dosages of50-100 μg, 100-300 μg, 300-400 μg, or 400-600 μg are administered everyother day. In alternate embodiments, dosages are calculated based onbody weight, and may be administered, for example, in amounts from about0.5 μg/m² to about 300 μg/m² per day, about 1 μg/m² to about 200 μg/m²,about 1 μg/m² to about 187.5 μg/m² per day, about 1 μg/m² per day toabout 175 μg/m² per day, about 1 μg/m² per day to about 157 μg/m² perday about 1 μg/m² to about 125 μg/m² per day, about 1 μg/m² to about 75μg/m² per day, 1 μg/m² to about 50/μg/m² per day, 2 μg/m² to about 50μg/m² per day, 2 μg/m² to about 30 μg/m² per day or 3 μg/m² to about 30μg/m² per day.

In other embodiments, dosages may be administered less frequently, forexample, 0.5 μg/m² to about 300 μg/m² every other day, about 1 μg/m² toabout 200 μg/m², about 1 μg/m² to about 187.5 μg/m² every other day,about 1 μg/m² to about 175 μg/m² every other day, about 1 μg/m² per dayto about 157 μg/m² every other day about 1 μg/m² to about 125 μg/m²every other day, about 1 μg/m² to about 75 μg/m² every other day, 1μg/m² to about 50 μg/m² every other day, 2 μg/m² to about 50 μg/m² everyother day, 2 μg/m² to about 30 μg/m² per day or 3 μg/m² to about 30μg/m² per day. In additional embodiments, dosages may be administered 3times/week, 4 times/week, 5 times/week, only on weekdays, only inconcert with other treatment regimens, on consecutive days, or in anyappropriate dosage regimen depending on clinical and patient-specificfactors.

The amount, timing and mode of delivery of compositions of the inventioncomprising a neoplastic disease treating effective amount of a phorbolester compound of Formula I or derivative compound of a phorbol ester ofFormula I, (Th1 cytokine increasing, ERK phosphorylation inducing, NFκBmodulating, chemotherapeutic, anti-tumor, cancer treating, remissioninducing, remission maintaining, apoptosis inducing effective amount) orchemoprotective or radioprotective effective amount of a phorbol esterof Formula I will be routinely adjusted on an individual basis,depending on such factors as weight, age, gender, and condition of theindividual, the acuteness of the cytopathic disease and/or relatedsymptoms, whether the administration is prophylactic or therapeutic, andon the basis of other factors known to effect drug delivery, absorption,pharmacokinetics, including half-life, and efficacy.

An effective dose or multi-dose treatment regimen for the instantdisease treating (alternatively, “Th1 cytokine increasing”, “ERKphosphorylation inducing”, ‘NFκB modulating,” “chemotherapeutic”,“anti-tumor”, “cancer treating”, “apoptosis inducing”, “remissioninducing”, “remission maintaining”, “chemoprotective,”“anti-inflammatory,” “neutrophil stimulating,” “erythropoiesisstimulating,” “bone resorbtion inhibiting,” “bone strengthening,”“antiemetic,” “pain relieving,” “radiation protective,” “anti-swelling,”“cytoprotective,” “anti-mucositis,” “epithelial stimulating,”“anti-fibrotic,” “platelet stimulating,”) formulations of the inventionwill ordinarily be selected to approximate a minimal dosing regimen thatis necessary and sufficient to substantially prevent or alleviate thesymptoms of the disease including cancer, in the subject, and/or tosubstantially prevent or alleviate one or more symptoms associated withneoplastic diseases such as cancer, or substantially prevent oralleviate one or more of the side effects of chemotherapy treatment orradiation treatment in the subject. A dosage and administration protocolwill often include repeated dosing therapy over a course of several daysor even one or more weeks or years. An effective treatment regime mayalso involve prophylactic dosage administered on a day or multi-dose perday basis lasting over the course of days, weeks, months or even years.

Various assays and model systems can be readily employed to determinethe therapeutic effectiveness of the treatment of cytopathic diseases.For example, effectiveness may be demonstrated using a complete bloodcount (CBC). The measurements taken in a CBC include a white blood cellcount (WBC), a red blood cell count (RBC), the red cell distributionwidth, the hematocrit, and the amount of hemoglobin. Some signs ofcancer, or responses to chemotherapy or radiation therapy which arevisible in a CBC include a low hematocrit, a sharp decrease in thenumber of blood platelets, and a low level of neutrophils. An effectiveamount of a composition of the present invention will increase thelevels measured in a complete blood count by 10%, 20%, 30%, 50% orgreater increase, up to a 75-90%, or 95% or greater. Effective amountswill also move the blood protein of an individual towards the optimalcategory for each type of protein.

Effectiveness in the treatment of neoplastic diseases may also bedetermined by a number of methods such as, but not limited to, ECOGPerformance Scale, the Karnofsky Performance Scale, microscopicexamination of blood cells, bone marrow aspiration and biopsy,cytogenetic analysis, biopsy, immunophenotyping, blood chemistrystudies, a complete blood count, lymph node biopsy, peripheral bloodsmear, visual analysis of a tumor or lesion, or any other method ofevaluating and/or diagnosing malignancies and tumor progression known tothose of skill in the art.

For example, effectiveness of the compositions and methods herein in thetreatment of hematologic malignancies/bone marrow disorders may beevaluated using, an absolute neutrophil count (ANC). A normal ANC isbetween 1,500 to 8,000/mm³. Individuals suffering from hematologicmalignancies/bone marrow disorders frequently have an ANC below1500/mm³, and may even reach levels below 500/mm³ Effective amounts ofthe compositions and methods herein will increase an individual's ANC by10%, 20%, 30%, 50% or greater increase, up to a 75-90%, or 95% orgreater. Effective amounts may increase ANC levels above 1500/mm³.

Effectiveness of the compositions and methods herein in the treatment ofhematologic malignancies/bone marrow disorders may further be evaluatedusing, for example, a platelet count. A platelet count is normallybetween 150,000 to 450,000 platelets per microliter (×10⁻⁶/Liter).Individuals suffering from hematologic malignancies/bone marrow disordermay have platelet counts below 100,000 per microliter. Effective amountsof the compositions and methods herein will increase an individual'splatelet count by 10%, 20%, 30%, 50% or greater increase, up to a75-90%, or 95% or greater. Effective amounts may increase plateletlevels above 100,000 per microliter.

Effectiveness of the compositions and methods herein in the treatment ofhematologic malignancies/bone marrow disorders may additionally beevaluated, for example, by measuring the number of myeloblasts.Myeloblasts normally represent less than 5% of the cells in the bonemarrow but should not be present in circulating blood. Effective amountsof the compositions and methods herein will decrease the number ofmyeloblasts by 10%, 20%, 30%, 50% or more, up to a 75-90%, 96% orgreater decrease. Effective amounts may decrease myeloblasts to below 5%of the cells in the bone marrow.

Effectiveness of the compositions and methods herein in the treatment ofhematologic malignancies/bone marrow disorders may further be evaluatedby examining myeloblasts for the presence of Auer rods. Effectiveamounts of the compositions of the present invention will decrease thenumber of Auer rods visible by 10%, 20%, 30%, 50% or more, up to a75-90%, 96% or greater decrease up to the complete elimination of Auerrods.

Effectiveness of the compositions and methods herein in the treatment ofneoplasms may be evaluated by screening for markers in the blood such asCA 15.3, TRU-QUANT, CA 27.29, CA125, CYFRA 21-1, ProGRP, CA19-9, CA242,CAM 17.1, tissue polypeptide specific antigen (TPS), serum macrophageinhibitory cytokine 1 (MIC-1, osteopontin, glypican-3 (GPC-3),des-γ-carboxy-prothrombin (DCP), α-Fetoprotein (AFP), tissue inhibitorof metalloproteinase type 1 (TIMP-1). squamous cell carcinoma antigen(SCCA), CEA (carcinoembryonic antigen), or circulating tumor cells.Effective amounts of the compositions of the present invention willdecrease the amount of the markers present by 10%, 20%, 30%, 50% ormore, up to a 75-90%, 96% or greater decrease up to the completeelimination of markers in the blood.

Effectiveness of the compositions and methods of the invention may alsobe demonstrated by a decrease in the symptoms of subjects suffering fromneoplastic disease including, but not limited to, anemia; chronicfatigue; excessive or easy bleeding, such as bleeding of the nose, gums,and under the skin; easy bruising, particularly bruising with noapparent cause; shortness of breath; petechiae; recurrent fever; swollengums; slow healing of cuts; bone and joint discomfort; recurrentinfections; weight loss; itching; night sweats; lymph node swelling;fever; abdominal pain and discomfort; disturbances in vision; coughing;loss of appetite; pain in the chest; difficulty swallowing; swelling ofthe face, neck and upper extremities; a need to urinate frequently,especially at night; difficulty starting urination or holding backurine; weak or interrupted flow of urine; painful or burning urination;difficulty in having an erection; painful ejaculation; blood in urine orsemen; frequent pain or stiffness in the lower back, hips, or upperthighs; and weakness.

Effectiveness of the compositions and methods of the invention may alsobe demonstrated by a decrease in the symptoms of chemotherapeutictreatment including, but not limited to, alopecia, nausea, vomiting,poor appetite, soreness, neutropenia, anemia, thrombocytopenia,dizziness, fatigue, constipation, oral ulcers, itchy skin, peeling,nerve and muscle leprosy, auditory changes, problems with blood, weightloss, diarrhea, immunosuppression, bruising, tendency to bleed easily,heart damage, liver damage, kidney damage, vertigo and encephalopathy.

Effectiveness of the compositions and methods of the invention may alsobe demonstrated by a decrease in the symptoms that accompany radiationtherapy including, but not limited to, moist desquamation, soreness,diarrhea, nausea, vomiting, appetite loss, constipation, itchy skin,peeling, mouth and throat sores, edema, infertility, fibrosis,epilation, and mucosal dryness in comparison to others who have receivedsimilar radiotherapy treatments.

For each of the indicated conditions described herein, test subjectswill exhibit a 10%, 20%, 30%, 50% or greater reduction, up to a 75-90%,or 96% or greater, reduction, in one or more symptom(s) caused by, orassociated with, the disease, or related diseases or conditions in thesubject, compared to placebo-treated or other suitable control subjects.

Within additional aspects of the invention, combinatorial diseasetreating (“Th1 cytokine increasing,” “ERK phosphorylation inducing,”“NFκB modulating”, “apoptosis inducing,” “chemotherapeutic,”“anti-tumor,” “cancer treating,” “remission inducing,” “remissionmaintaining,” “chemoprotective,” “anti-inflammatory,” “neutrophilstimulating,” “erythropoiesis stimulating,” “bone resorbtioninhibiting,” “bone strengthening,” “antiemetic,” “pain relieving,”“radiation protective,” “anti-swelling,” “cytoprotective,”“anti-mucositis,” “epithelial stimulating,” “anti-fibrotic,” “plateletstimulating,”) formulations and coordinate administration methods areprovided which employ an effective amount of a phorbol ester compound ofFormula I or a derivative compound of Formula I and one or moresecondary or adjunctive agent(s) that is/are combinatorially formulatedor coordinately administered with the phorbol ester compound of FormulaI to yield a combined, multi-active disease treating composition orcoordinate treatment method.

Exemplary combinatorial formulations and coordinate treatment methods inthis context employ the phorbol ester of Formula I or derivativecompound of a phorbol ester of Formula I, in combination with one ormore secondary anti-tumor agent(s), or with one or more adjunctivetherapeutic agent(s) that is/are useful for treatment or prophylaxis ofthe targeted (or associated) disease, condition and/or symptom(s) in theselected combinatorial formulation or coordinate treatment regimen. Formost combinatorial formulations and coordinate treatment methods of theinvention, a phorbol ester compound of Formula I or related orderivative compound is formulated, or coordinately administered, incombination with one or more secondary or adjunctive therapeuticagent(s), to yield a combined formulation or coordinate treatment methodthat is combinatorially effective or coordinately useful to treatneoplastic diseases and one or more symptom(s) of a secondary disease orcondition in the subject. Exemplary combinatorial formulations andcoordinate treatment methods in this context employ a phorbol estercompound of Formula I, or derivative compound of a phorbol ester ofFormula I, in combination with one or more secondary or adjunctivetherapeutic agents selected from, e.g., chemotherapeutic agents,anti-inflammatory agents, doxorubicin, vitamin D3, cytarabine, cytosinearabinoside, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin,idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin,asparaginase, carboplatin, etoposide phosphate, fludarabine,methotrexate, etoposide, dexamethasone, and choline magnesiumtrisalicylate. In addition, adjunctive or secondary therapies may beused such as, but not limited to, radiation treatment, hormone therapyand surgery.

Exemplary combinatorial formulations and coordinate treatment methods inthe prevention or treatment of side effects from chemotherapy employ thephorbol ester compound of Formula I, or derivative compound of a phorbolester of Formula I, in combination with one or more additional,chemoprotective or other indicated, secondary or adjunctive therapeuticagents that is/are useful for treatment or prophylaxis of the targeteddisease, condition and/or symptom(s). For most combinatorialformulations and coordinate treatment methods of the invention, aphorbol ester compound of Formula I or related or derivative compound isformulated, or coordinately administered, in combination with one ormore secondary or adjunctive therapeutic agent(s), to yield a combinedformulation or coordinate treatment method that is combinatoriallyeffective or coordinately useful to prevent or treat side effects ofchemotherapy in the subject. Exemplary combinatorial formulations andcoordinate treatment methods in this context employ a phorbol estercompound of Formula I in combination with one or more secondary oradjunctive therapeutic agents selected from, pegfilgrastim, epoeitnalfa, darbepoetin alfa, alendronate sodium, risedronate, ibandronate,G-CSF, 5-HT₃ receptor antagonists, NK₁ antagonists, olanzapine,corticosteroids, dopamine antagonists, serotonin antagonists,benzodiazepines, aprepitant, and cannabinoids.

Exemplary combinatorial formulations and coordinate treatment methods inthe prevention or treatment of side effects from radiation therapyemploy the phorbol ester compound of Formula I or a derivative compoundof Formula I in combination with one or more additional, radioprotectiveor other indicated secondary or adjunctive therapeutic agents thatis/are useful for treatment or prophylaxis of the targeted conditionand/or symptom(s). For most combinatorial formulations and coordinatetreatment methods of the invention, a phorbol ester compound of FormulaI or related or derivative compound is formulated, or coordinatelyadministered, in combination with one or more secondary or adjunctivetherapeutic agent(s), to yield a combined formulation or coordinatetreatment method that is combinatorially effective or coordinatelyuseful to prevent or treat side effects of radiation therapy in thesubject. Exemplary combinatorial formulations and coordinate treatmentmethods in this context employ a phorbol ester compound of Formula I, orderivative compound of a phorbol ester of Formula I, in combination withone or more secondary or adjunctive therapeutic agents selected fromsteroids, amifostine, chlorhexidine, benzydamine, sucralfate,keratinocyte growth factor (KGF), palifermin, Cu/Zn superoxidedismutase, Interleukin 11, or prostaglandins.

In certain embodiments the invention provides combinatorial diseasetreating (“Th1 cytokine increasing,” “ERK phosphorylation inducing,”“NFκB modulating,” “apoptosis inducing,” “chemotherapeutic,”“anti-tumor,” “cancer treating,” “remission inducing,” “remissionmaintaining,” “chemoprotective,” “anti-inflammatory,” “neutrophilstimulating,” “erythropoiesis stimulating,” “bone resorbtioninhibiting,” “bone strengthening,” “antiemetic,” “pain relieving,”“radiation protective,” “anti-swelling,” “cytoprotective,”“anti-mucositis,” “epithelial stimulating,” “anti-fibrotic,” “plateletstimulating,”) formulations comprising a phorbol ester and one or moreadjunctive agent(s) having disease treating activity. Within suchcombinatorial formulations, a phorbol ester of Formula I and theadjunctive agent(s) having disease treating activity will be present ina combined formulation in disease treating (“Th1 cytokine increasing,”“ERK phosphorylation inducing,” “NFκB modulating”, “apoptosis inducing,”“chemotherapeutic,” “anti-tumor,” “cancer treating,” “remissioninducing,” “remission maintaining,” “chemoprotective,”“anti-inflammatory,” “neutrophil stimulating,” “erythropoiesisstimulating,” “bone resorbtion inhibiting,” “bone strengthening,”“antiemetic,” “pain relieving,” “radiation protective,” “anti-swelling,”“cytoprotective,” “anti-mucositis,” “epithelial stimulating,”“anti-fibrotic,” “platelet stimulating,”) effective amounts, alone or incombination. In exemplary embodiments, a phorbol ester compound ofFormula I and a non-phorbol ester agent(s) will each be present in adisease treating/preventing amount (i.e., in singular dosage which willalone elicit a detectable alleviation of symptoms in the subject).Alternatively, the combinatorial formulation may comprise one or boththe phorbol ester compound of Formula I and the non-phorbol ester agentsin sub-therapeutic singular dosage amount(s), wherein the combinatorialformulation comprising both agents features a combined dosage of bothagents that is collectively effective in eliciting a neoplastic diseaseor symptom alleviating response or a radioprotective or chemoprotectiveresponse. Thus, one or both of the phorbol ester of Formula I, orderivative compound of a phorbol ester of Formula I, and non-phorbolester agents may be present in the formulation, or administered in acoordinate administration protocol, at a sub-therapeutic dose, butcollectively in the formulation or method they elicit a detectabledecrease in neoplasms, symptoms of neoplastic disease or side effectsfrom chemotherapy or radiation treatments of neoplastic diseases in thesubject. For example, in some embodiments, the combinatorial formulationmay include one or more additional chemotherapeutic agents. In a furtherembodiment, the combinatorial formulation may include one or moreadditional chemoprotective agents. In other embodiments, thecombinatorial formulation may include one or more radioprotectiveagents. In a further embodiment, the combinatorial formulation mayinclude one or more anti-inflammatory agents or other secondary oradditional therapeutic agents as described herein.

To practice coordinate administration methods of the invention, aphorbol ester compound of Formula I, or derivative compound of a phorbolester of Formula I, may be administered, simultaneously or sequentially,in a coordinate treatment protocol with one or more of the secondary oradjunctive therapeutic agents contemplated herein. Thus, in certainembodiments a compound is administered coordinately with a non-phorbolester agent, or any other secondary or adjunctive therapeutic agentcontemplated herein, using separate formulations or a combinatorialformulation as described above (i.e., comprising both a phorbol estercompound of Formula I or related or derivative compound, and anon-phorbol ester therapeutic agent). This coordinate administration maybe done simultaneously or sequentially in either order, and there may bea time period while only one or both (or all) active therapeutic agentsindividually and/or collectively exert their biological activities.

In another embodiment, such coordinate treatment methods may, forexample, follow or be derived from various chemotherapeutic protocols.Other coordinate treatment methods may, for example, include a phorbolester and/or treatments for additional symptoms of neoplastic diseases.A distinguishing aspect of all such coordinate treatment methods is thatthe phorbol ester compound of Formula I, or derivative compound of aphorbol ester of Formula I, exerts at least some activity, which yieldsa favorable clinical response in conjunction with a complementaryneoplastic disease symptom decreasing, or distinct, clinical responseprovided by the secondary or adjunctive therapeutic agent. Often, thecoordinate administration of the phorbol ester compound of Formula I orderivative compound of a phorbol ester of Formula I, with the secondaryor adjunctive therapeutic agent will yield improved therapeutic orprophylactic results in the subject beyond a therapeutic effect elicitedby the phorbol ester compound of Formula I or derivative compound of aphorbol ester of Formula I, or the secondary or adjunctive therapeuticagent administered alone. This qualification contemplates both directeffects as well as indirect effects.

Within exemplary embodiments, a phorbol ester compound of Formula I orderivative compound of a phorbol ester of Formula I will be coordinatelyadministered (simultaneously or sequentially, in combined or separateformulation(s)), with one or more secondary cancer treating agents, orother indicated or adjunctive therapeutic agents, e.g. doxorubicin,vitamin D3, cytarabine, cytosine arabinoside, daunorubicin,cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine,mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin,etoposide phosphate, fludarabine, methotrexate, etoposide,dexamethasone, and choline magnesium trisalicylate.

In another embodiment, such coordinate treatment methods may, forexample, follow or be derived from various palliative protocols forchemotherapy patients. Coordinate treatment methods may, for example,include a phorbol ester and/or treatments for additional side effects ofchemotherapy. A distinguishing aspect of all such coordinate treatmentmethods is that the phorbol ester compound of Formula I or derivativecompound of a phorbol ester of Formula I, exerts at least some activity,which yields a favorable clinical response in conjunction with acomplementary chemotherapeutic side effect alleviating, or distinct,clinical response provided by the secondary or adjunctive therapeuticagent. Often, the coordinate administration of the phorbol estercompound of Formula I, or derivative compound of a phorbol ester ofFormula I, with the secondary or adjunctive therapeutic agent will yieldimproved therapeutic or prophylactic results in the subject beyond atherapeutic effect elicited by the phorbol ester compound of Formula I,or derivative compound of a phorbol ester of Formula I, or the secondaryor adjunctive therapeutic agent administered alone. This qualificationcontemplates both direct effects as well as indirect effects.

Within exemplary embodiments, a phorbol ester compound of Formula I, orderivative compound of a phorbol ester of Formula I, will becoordinately administered (simultaneously or sequentially, in combinedor separate formulation(s)), with one or more secondary chemotherapeuticside affect alleviating compounds or other indicated or adjunctivetherapeutic agents, e.g. pegfilgrastim, epoeitn alfa, darbepoetin alfa,alendronate sodium, risedronate, ibandronate, G-CSF, 5-HT₃ receptorantagonists, NK₁ antagonists, olanzapine, corticosteroids, dopamineantagonists, serotonin antagonists, benzodiazepines, aprepitant, andcannabinoids.

In another embodiment, such coordinate treatment methods may, forexample, follow or be derived from various palliative protocols forradiation therapy patients. Coordinate treatment methods may, forexample, include a phorbol ester and/or treatments for additional sideeffects of radiation therapy. A distinguishing aspect of all suchcoordinate treatment methods is that the phorbol ester compound ofFormula I or derivative compound of a phorbol ester of Formula I, exertsat least some activity, which yields a favorable clinical response inconjunction with a complementary radiotherapy side effect alleviating,or distinct, clinical response provided by the secondary or adjunctivetherapeutic agent. Often, the coordinate administration of the phorbolester compound of Formula I or derivative compound of a phorbol ester ofFormula I with the secondary or adjunctive therapeutic agent will yieldimproved therapeutic or prophylactic results in the subject beyond atherapeutic effect elicited by the phorbol ester compound of Formula Ior derivative compound of a phorbol ester of Formula I, or the secondaryor adjunctive therapeutic agent administered alone. This qualificationcontemplates both direct effects as well as indirect effects.

Within exemplary embodiments, a phorbol ester compound of Formula I orderivative compound of a phorbol ester of Formula I, will becoordinately administered (simultaneously or sequentially, in combinedor separate formulation(s)), with one or more secondary radiotherapyside affect alleviating compounds or other indicated or adjunctivetherapeutic agents, e.g. steroids, amifostine, chlorhexidine,benzydamine, sucralfate, keratinocyte growth factor (KGF), palifermin,Cu/Zn superoxide dismutase, Interleukin 11, or prostaglandins.

As noted above, in all of the various embodiments of the inventioncontemplated herein, the disease treating methods and formulations mayemploy a phorbol ester compound of Formula I in any of a variety offorms, including any one or combination of the subject compound'spharmaceutically acceptable salts, solvates, isomers, enantiomers,polymorphs, solvates, hydrates, and/or prodrugs. In exemplaryembodiments of the invention, TPA is employed within the therapeuticformulations and methods for illustrative purposes.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended therapeutic orprophylactic purpose. Suitable routes of administration for thecompositions of the invention include, but are not limited to,conventional delivery routes, devices and methods including injectablemethods such as, but not limited to, intravenous, intramuscular,intraperitoneal, intraspinal, intrathecal, intracerebroventricular,intraarterial, subcutaneous and intranasal routes.

The compositions of the present invention may further include apharmaceutically acceptable carrier appropriate for the particular modeof administration being employed. Dosage forms of the compositions ofthe present invention include excipients recognized in the art ofpharmaceutical compounding as being suitable for the preparation ofdosage units as discussed above. Such excipients include, withoutintended limitation, binders, fillers, lubricants, emulsifiers,suspending agents, sweeteners, flavorings, preservatives, buffers,wetting agents, disintegrants, effervescent agents and otherconventional excipients and additives.

If desired, the compositions of the invention can be administered in acontrolled release form by use of a slow release carrier, such as ahydrophilic, slow release polymer. Exemplary controlled release agentsin this context include, but are not limited to, hydroxypropyl methylcellulose, having a viscosity in the range of about 100 cps to about100,000 cps or other biocompatible matrices such as cholesterol.

Some phorbol ester compositions of Formula I of the invention aredesigned for parenteral administration, e.g. to be administeredintravenously, intramuscularly, subcutaneously or intraperitoneally,including aqueous and non-aqueous sterile injectable solutions which,like many other contemplated compositions of the invention, mayoptionally contain anti-oxidants, buffers, bacteriostats and/or soluteswhich render the formulation isotonic with the blood of the mammaliansubject; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and/or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers. Additionalcompositions and formulations of the invention may include polymers forextended release following parenteral administration. The parenteralpreparations may be solutions, dispersions or emulsions suitable forsuch administration. The subject agents may also be formulated intopolymers for extended release following parenteral administration.Pharmaceutically acceptable formulations and ingredients will typicallybe sterile or readily sterilizable, biologically inert, and easilyadministered. Such polymeric materials are well known to those ofordinary skill in the pharmaceutical compounding arts. Parenteralpreparations typically contain buffering agents and preservatives, andinjectable fluids that are pharmaceutically and physiologicallyacceptable such as water, physiological saline, balanced salt solutions,aqueous dextrose, glycerol or the like. Extemporaneous injectionsolutions, emulsions and suspensions may be prepared from sterilepowders, granules and tablets of the kind previously described.Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as described herein above, or an appropriatefraction thereof, of the active ingredient(s).

In more detailed embodiments, compositions of the invention may comprisea phorbol ester compound of Formula I or derivative compound of aphorbol ester of Formula I encapsulated for delivery in microcapsules,microparticles, or microspheres, prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively; in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules); or withinmacroemulsions.

As noted above, in certain embodiments the methods and compositions ofthe invention may employ pharmaceutically acceptable salts, e.g., acidaddition or base salts of the above-described phorbol ester compounds ofFormula I and/or related or derivative compounds. Examples ofpharmaceutically acceptable addition salts include inorganic and organicacid addition salts. Suitable acid addition salts are formed from acidswhich form non-toxic salts, for example, hydrochloride, hydrobromide,hydroiodide, sulphate, hydrogen sulphate, nitrate, phosphate, andhydrogen phosphate salts. Additional pharmaceutically acceptable saltsinclude, but are not limited to, metal salts such as sodium salts,potassium salts, cesium salts and the like; alkaline earth metals suchas calcium salts, magnesium salts and the like; organic amine salts suchas triethylamine salts, pyridine salts, picoline salts, ethanolaminesalts, triethanolamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts and the like; organic acid salts suchas acetate, citrate, lactate, succinate, tartrate, maleate, fumarate,mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, andformate salts; sulfonates such as methanesulfonate, benzenesulfonate,and p-toluenesulfonate salts; and amino acid salts such as arginate,asparaginate, glutamate, tartrate, and gluconate salts. Suitable basesalts are formed from bases that form non-toxic salts, for examplealuminum, calcium, lithium, magnesium, potassium, sodium, zinc anddiethanolamine salts.

Other detailed embodiments, the methods and compositions of theinvention for employ prodrugs of phorbol esters of Formula I. Prodrugsare considered to be any covalently bonded carriers which release theactive parent drug in vivo. Examples of prodrugs useful within theinvention include esters or amides with hydroxyalkyl or aminoalkyl as asubstituent, and these may be prepared by reacting such compounds asdescribed above with anhydrides such as succinic anhydride.

The invention disclosed herein will also be understood to encompassmethods and compositions comprising phorbol esters of Formula I using invivo metabolic products of the said compounds (either generated in vivoafter administration of the subject precursor compound, or directlyadministered in the form of the metabolic product itself). Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification and the like of the administered compound,primarily due to enzymatic processes. Accordingly, the inventionincludes methods and compositions of the invention employing compoundsproduced by a process comprising contacting a phorbol ester compound ofFormula I with a mammalian subject for a period of time sufficient toyield a metabolic product thereof. Such products typically areidentified by preparing a radiolabelled compound of the invention,administering it parenterally in a detectable dose to an animal such asrat, mouse, guinea pig, monkey, or to man, allowing sufficient time formetabolism to occur and isolating its conversion products from theurine, blood or other biological samples.

The invention disclosed herein will also be understood to encompassdiagnostic compositions for diagnosing the risk level, presence,severity, or treatment indicia of, or otherwise managing diseasesincluding, but not limited to, neoplastic diseases including malignantneoplastic diseases such as leukemia, or a related disease or conditionin a mammalian subject, comprising contacting a labeled (e.g.,isotopically labeled, fluorescent labeled or otherwise labeled to permitdetection of the labeled compound using conventional methods) phorbolester compound of Formula I to a mammalian subject (e.g., to a cell,tissue, organ, or individual) at risk or presenting with one or moresymptom(s) of cancer, and thereafter detecting the presence, location,metabolism, and/or binding state (e.g., detecting binding to anunlabeled binding partner involved in malignant cell receptorphysiology/metabolism) of the labeled compound using any of a broadarray of known assays and labeling/detection methods. In exemplaryembodiments, a phorbol ester compound of Formula I isisotopically-labeled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl,respectively. The isotopically-labeled compound is then administered toan individual or other subject and subsequently detected as describedabove, yielding useful diagnostic and/or therapeutic management data,according to conventional techniques.

EXAMPLES

The experiments described below demonstrate novel and powerful uses forphorbol esters and related derivative compounds in the treatment ofneoplastic diseases, as well as their activity as chemoprotectants andradioprotectants. These and additional findings are further expanded andelucidated within the following examples.

Example I Effect of TPA on the Peripheral White Blood Cells (WBC) andHemoglobin (Hb) Counts in S180 Cell-Injected Mice

Sarcoma 180 (S180) cells were injected into Kwen-Ming mice. On the thirdday, the mice were given TPA interperitoneally (i.p.). at 50, 100 or 200μg/kg/day for 7 days. On the second day after the treatment wascompleted, blood samples were taken from the tails of the treated micefor WBC and Hb analyses. The WBC counts for the treated groups (50, 100,or 200 ug/kg/day for 7 days) were 16.1±7.4, 18.7±0.3.0 and20.7±0.3.4×10⁹/L, respectively; the WBC count for the control group was13.6±1.8×10⁹/L. The Hb of the treated groups were 136±11, 149±12 and149±10 g/L, and the Hb of the control group was 134+−15 g/L. The resultsindicate that i.p. injection of TPA could increase the peripheral WBCcounts in mice in a dose-dependent manner, whereas the Hb levels werenot greatly affected in TPA treated mice when compared to the controlmice.

Example II Dose Ranging Study

Due to the strong local irritation caused by TPA application, TPA wasgiven to patients by intravenous (i.v.) infusion. TPA solution in asterile syringe was injected into 200 ml of sterile saline and mixedwell for i.v. infusion.

The Toxicity and Side Effects of Different TPA Doses AdministeredClinically:

(1) TPA Given at 1 mg/Patient/Week:

One mg TPA in solution was mixed well with 200 ml of sterile saline forintravenous infusion which was completed in 1 h at the rate of 16μg/min. One hour after TPA administration, patients started to havechills which lasted for about 30 min, followed by fever, (the patients'temperature reached 37.5-39.5° C. which lasted for 3-5 h, then returnedto normal) with light to heavy perspiration. The above symptoms could bealleviated by giving the patients glucocorticoids. TPA at this dosecaused a minority of patients to bleed, several patients suffered for ashort period of time difficulty in breathing, and Hb was detected in theurine. However, these side effects were short lived and reversible. Thecardiac, hepatic, renal and pulmonary functions were all found to benormal.

(2) TPA Given at 0.5 mg/Patient×2/Week: (Two Doses a Week)

0.5 mg of TPA in solution was mixed well with 200 ml of saline forintravenous infusion which was completed in 1 h at the rate of 8 μg/min.The reactions after administration were similar to that of the 1 mg TPAdosage, but to a lesser extent than the 1 mg dose. The patientstolerated the lower dose more easily. Occasionally, Hb was detected inpatients' urine. Difficulty in breathing was not observed. The cardiac,hepatic, renal and pulmonary functions were all normal.

(3) TPA Given at 0.25 mg/Patient×4/Week:

0.25 mg of TPA in solution was mixed well with 200 ml of saline forintravenous infusion which was completed in 1 h at the rate of 4 μg/min.After administration, symptoms such as chills and fever were alsoobserved, but to a much lesser extent than with the higher dosages. NoHb was detected in the urine, and no patient suffered difficulty inbreathing. The cardiac, hepatic, renal and pulmonary functions were allnormal.

Example III Treatment of Relapsed/Refractory Malignancies with TPA

Patients with histologically documented relapsed/refractory hematologicmalignancy/bone marrow disorders are treated with a combination of TPA(Xichuan Pharmaceuticals, Nan Yang, Henan, China), dexamethasone andcholine magnesium trisalicylate. Comparable methods as set forth belowfor demonstrating the therapeutic use of TPA in the treatment of AcuteMyelogenous Leukemia (AML) will be applied to demonstrate the use of TPAfor treating other neoplastic conditions and malignancies. Otherneoplastic conditions and malignant disorders amenable to treatmentusing the methods and compositions of the invention include variousforms of cancer, including blood and bone malignancies and solid tumorsof various types. In addition to the specific protocols herein,successful treatment and/or remission will be determined for differenttargeted neoplastic and malignant conditions using any of a wide varietyof well known cancer detection and assessment methods—for example bydetermining size reduction of solid tumors, histopathological studies toevaluate tumor growth, stage, metastatic potential, presence/expressionlevels of histological cancer markers, etc.

AML is an aggressive disease that generally warrants urgent andintensive therapy. The average patient age at AML diagnosis is 64-68years old, and patients over the age of 60 treated with standardchemotherapy are cured of their disease <20% of the time. Patients whodevelop AML after an antecedent hematologic disorder or priorleukemogenic chemotherapy/radiation therapy have similarly pooroutcomes, as do patients whose disease is associated with specificadverse cytogenetic and clinical features. Hence, most patientsdiagnosed with AML have patient and/or disease-related features that areassociated with a very poor prognosis. For patients with relapseddisease, no standard non-transplant therapy has demonstrated thecapacity for cure. For these patients, AML is often a fatal disease. Newapproaches to the therapy of AML are needed.

Employing the methods and compositions of the instant invention, TPA, isdeveloped as a therapeutic agent for treating patients with AML, basedon TPA's novel role in modulating intracellular signaling pathways, it'scapacity to induce differentiation and/or apoptosis in cell lines, andclinical data indicating the effectiveness of TPA in treating neoplasticand malignant disorders, including myeloid malignancies.

Thus far clinical evaluation of TPA has demonstrated that TPA exertsdirect therapeutic cytotoxic effects in at least a subset of AML cases,as measured by cell viability and apoptosis assays. In all primarycultures analyzed by Western analysis, TPA strongly induced ERKphosphorylation by 1 hour in culture. TPA's cytotoxic effect on primaryAML cells is associated with the subsequent loss of the phospho-ERKpro-survival signal after 24 hour ex vivo exposure. This observation isin good agreement with other studies that reported decreased primary AMLsurvival after pharmacological interruption of ERK signaling by MEKinhibitors, such as PD98059, U0126 and PD 184352. In our studies, lossof ERK signaling was associated with induction of ERK phosphatases.

In addition to protein kinase C and ERK activation, TPA is a knowninducer of NF-κB, a pro-survival transcription factor oftenconstitutively active in AML blasts and leukemic stem cells. Recent workfrom our laboratory has demonstrated that AML cell NF-κB can beinhibited in vivo with 48 h of treatment with dexamethasone+cholinemagnesium trisalicylate (CMT). In addition, we have shown thatdexamethasone can induce MKP-1 ERK phosphatase expression and enhanceTPA cytotoxicity on primary AML samples. In this context, we have chosenin exemplary embodiments below to use dexamethasone and CMT asadjunctive medications to be used 24 h pre- and 24 h post treatment withTPA. These medications are well-tolerated and anticipated to reduceinflammatory adverse effects of treatment and enhance TPA cytotoxicityby increasing ERK phosphatase expression and inhibiting NF-κB. Inaddition dexamethasone and CMT will be used as adjunctive medicationsbecause they are anti-inflammatory, may ameliorate adverse effects, andmay enhance anti-leukemic activity by inhibition of the anti-apoptoticeffects of constitutive NF-κB expression and induction of phosphatasesthat decrease signaling pathway activity.

An initial TPA Phase 1 study enrolled 35 patients [23 withrelapsed/refractory AML, 2 with other myeloid malignancies (CML-blastcrisis, myelodysplasia with excess blasts), 3 with Hodgkin's Disease, 3with non-Hodgkin's lymphoma and 4 with solid tumors]. The majority ofpatients had relapsed/refractory AML. Our clinical results include oneAML patient with stable disease for >5 months, who received 8 TPAinfusions. In a second AML patient, a pronounced (5-fold) decline in thenumber of circulating blasts was seen following TPA administration. Thisdecline in leukemic blasts persisted for 4 weeks, and the patienteventually died from a fungal infection. Finally, a patient withrelapsed and refractory Hodgkin's disease despite high dose chemotherapywith autologous stem cell rescue had a partial remission of a chest wallmass after TPA administration. TPA dose escalation has been completed,in the last cohort 2 out of 3 patients treated at a dose of 0.188 mg/m2d1-5, 8-12 experienced grade III non-hematologic dose limitingtoxicities (DLT), establishing the maximum tolerated TPA dose as asingle agent at 0.125 mg/m2/d on d1-5 and 8-12.

In the case of AML and other hematologic malignancies, patients aregiven an initial dose of TPA of 1 mg/week×3 weeks (days 1, 8, 15)administered with continuous/intermittent pulse oximetry for 6 hours.Twenty four hours prior to initiation of TPA therapy, patients are given10 mg of dexamethasone every six hours and 1500 mg of choline magnesiumtrisalicylate (CMT) every eight hours continuing until 24 hours afteradministration of TPA. After administration of the initial dose of TPA,patients have a two week rest period after which they may bereevaluated. Those patients that have a disease response orstabilization from the initial dose of TPA are treated for up to sixcycles of twenty-eight days according to the protocol below.

Following the two week rest period, patients are pre-medicated withTylenol 650 mg and Benadryl 25-50 mg (depending on the patient's sizeand age) thirty minutes prior to administration of TPA. They are thengiven an intravenous infusion of TPA through a central venous catheterdaily for 5 days a week for two consecutive weeks followed by a 2-weekrest period. TPA is administered at a dose of 1 mg in 200 ml of normalsaline over 1 hour. Twenty four hours prior to initiation of TPAtherapy, patients are given 10 mg of dexamethasone every six hours and1500 mg of choline magnesium trisalicylate continuing every eight hoursuntil 24 hours after administration of the TPA.

Blood levels of TPA are measured prior to and after infusion using abioassay that measures organic solvent extractable differentiationactivity. 1 ml of blood is extracted twice with 5 ml of ethyl acetate,redissolving the extraction residue in 50 μL of ethanol and addition ofan aliquot of HL60 cells. After 48 hours, adherent cells are measured.

Tests are also run on blood samples taken prior to and after infusionwith TPA to determine levels of white blood cells, platelets, andneutrophils. The samples are additionally analyzed for the presence ofmyeloblasts and Auer rods. These and continuing experiments will furtherelucidate the therapeutic cytotoxic and other effects that TPA elicitsagainst neoplastic cells in AML and other neoplastic and malignantconditions.

Example IV Measurement of the Modulation of ERK Activation

Phospho-ERK levels are measured in circulating malignant cells inpatients with leukemia and in peripheral blood mononuclear cells inlymphoma/solid tumor patients. A blood sample is taken from patientstreated according to the protocol of Example III both prior to and afteradministration of TPA.

In leukemia patients with a WBC≥1000 per μL, flow cytometry is performedon a blood sample using cell surface antigen-specific and phospho-ERKspecific antibodies directly conjugated to fluorophores (BD Biosciences,San Jose, Calif.). Samples are taken pre-administration of TPA and onehour after infusion of TPA on days 1, 2, and 11 in the initial treatmentaccording to the protocol of Example III and days 1 and 11 in subsequentcycles. In leukemia patients with an absolute leukemic blast number≥2500 per μL and other non-leukemic patients, peripheral blood samplesare taken on days 1, 8 and 15 of the initial cycle according to theprotocol of Example III prior to and 1 and 4 hours post infusion.Samples are also analyzed using Western blot analysis for phosphor-ERK,and total ERK1/2 levels to confirm the results obtained from the flowcytometry and correlated to clinical responses.

The foregoing analyses will further elucidate TPA's role in treatment ofneoplastic and malignant conditions, including TPA's cytotoxic effect onmalignant cells, exemplified by primary AML cells, and the associatedreduction by TPA of the phosphor-ERK pro-survival signal.

Example V Measurement of NF-κB Modulation

In prior studies we have shown that NF-κB activity can be modulated inpatients following administration of TPA with dexamethasone.Additionally, dexamethasone has been shown to induce MKP-1 ERKphosphatase expression and enhance TPA cytotoxicity. The followingstudies are designed to further elucidate how NF-κB activity istherapeutically modulated in patients treated with TPA plusdexamethasone.

NF-κB binding is measured in patient peripheral blood samples atbaseline and pre and post infusion from patients treated with TPAaccording to Example III using ELISA-based assays (BD Bioscience, SanJose, USA). NF-κB levels are quantified using chemiluminescent intensityto detect bingeing in limiting amounts of cellular extract using a96-well format. Additionally, electrophoretic mobility shift assays areperformed to measure NF-κB binding in peripheral blood samples fromleukemia patient with an absolute leukemic blast number ≥2500 per μL andother non-leukemic patients with normal white blood cell counts.

The foregoing studies will further show that TPA is an inducer of NF-κB,however these experiments demonstrate that AML cell NF-κB can beinhibited with treatment with dexamethasone and choline magnesiumtrisalicylate.

Example VI Determination of Changes in Leukemic Gene Expression

TPA induces RNA levels of several dual specificity phosphatases capableof terminating pro-survival ERK pathway signaling. A blood sample takenpre- and post-infusion from patients with AML treated with TPA accordingto Example III is used to study RNA expression of AML signalingcomponents such as the MAPK-specific DUSPs using quantitative realtimeRT-PCR and oligonucleotide microarray analysis.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications may be practicedwithin the scope of the appended claims which are presented by way ofillustration not limitation. In this context, various publications andother references have been cited with the foregoing disclosure foreconomy of description. Each of these references is incorporated hereinby reference in its entirety for all purposes. It is noted, however,that the various publications discussed herein are incorporated solelyfor their disclosure prior to the filing date of the presentapplication, and the inventors reserve the right to antedate suchdisclosure by virtue of prior invention.

Example VII Treatment of Lymphoma

Patient M. J., age 60, male, was diagnosed with a re-occurrence oflymphoma and a mass of 3.5 cm in diameter. The patient was given 15injections of 0.19 mg of TPA (0.125 mg/m²) every other day for 30 daysand the mass disappeared. As of 2011, he has been in remission for threeyears.

Example VIII Treatment of Breast Cancer

Patient M. L., female, age 50, was diagnosed with terminal breastcancer. She was unresponsive to either radiation or chemotherapy and thecancer had metastasized into the bone leaving her wheelchair bound. Shereceived 35 injections of TPA with a progressing dose from 0.18 mg ofTPA (1×0.125 mg/m²) to 0.26 mg of TPA (1.5×0.125 mg/m²), three to fourtimes, a week and is now in remission and able to walk normally.

Example IX Treatment of Lung Cancer

Patient J. L., male, age 56, was diagnosed with terminal lung cancerwhich was refractory to chemotherapy. The cancer metastasized into hisbones leaving him unable to walk. After 35 injections of TPA with aprogressing dose from 0.19 mg of TPA (1×0.125 mg/m²) to 0.26 mg of TPA(1.5×0.125 mg/m²) three to four times a week, he is in remission andable to walk normally.

Example X Treatment of Liver Cancer

Patient X, male, age ? was diagnosed with metastatic liver cancer. Hisinitial alpha fetoprotein level was 48,813. He was given chemotherapyand radiation treatments but his alpha fetoprotein level remainedelevated at 50,000+. He then received three injections of 0.19 mg of TPA(0.125 mg/m²) and his alpha fetoprotein levels began dropping andreturned to normal levels within four months.

Example XI TPA As an Adjuvant to Traditional Neoplasm Treatments

Patient N. K., female, 54, was diagnosed with terminal metastasizedpancreatic cancer. She received five injections of 0.18 mg TPA (0.125mg/m²) per week for 12 weeks in addition to chemotherapy. Her treatmentreduced the tumor in the pancreas from 6.3 cm to 2.4 cm. The patientmaintained her appetite, did not lose her hair and had significantlyless vomiting and nausea than in prior chemotherapy treatments withoutTPA.

Patient P. T., male, 42, was diagnosed with non-small-cell lung cancer.The cancer had metastasized and was refractory to Tarceva® (erlotinib)and Iressa™ (gefitinib). The patient was treated with a combination ofgemcitabine and cisplatin according to standard protocols accompanied byan injection of 0.19 mg of TPA (0.125 mg/m²) each weekday for eightweeks. During the combined chemotherapy and TPA treatment he did notlose any hair and had significantly less nausea than experienced duringprior chemotherapy treatments. He has been in remission since Jun. 30,2010.

Patient B. L., male, age 59, was diagnosed with terminal nasopharyngealcarcinoma and treated with both chemotherapy and radiotherapy. Hereceived injections of 0.19 mg of TPA (0.125 mg/m²) of TPA a day forfive days prior to beginning radiotherapy and then 0.19 mg of TPA (0.125mg/m²) every other day for a total of 20 injections. He has been inremission for two and a half years and did not suffer any apparent skindamage from the radiation treatment.

Example XII Chemoprotective Effect of TPA

A colony formation assay including semi-solid medium formulated withDMEM and 0.5% agar is used. For these cultures, mononuclear cells areplated at a concentration of about 2.5×10⁵ cells/mL and GM-CSF and G-CSFare added at a concentration of about 100 U/mL. Cells are cultured for14 days in a 5% CO₂ incubator, with 100% humidity at 37. C. At the endof the culture period, colonies of 50 or more cells are counted using aninverted microscope by two independent viewers. (Hamburger, 1977)

Peripheral stem cells are randomized into 4 groups at a concentration of5×10⁵ cells/mL in DMEM supplemented with 10% fetal calf serum. Groups 1and 4 are untreated control and groups 2 and 3 are incubated for 24hours with 0.05 μg/mL of TPA. After 24 hours, cells are washed with DMEM10% fetal calf serum. Groups 3 and 4 are then incubated with 25 μg/mL of5-fluorodeoxyuridine monophosphate, the metabolite of fluorouracil, for20 hours. Subsequently, all groups are washed twice and the cells areplated in semi-solid agar medium. The colonies are counted at 14 days.

Example XIII Use of TPA to Protect Against Radiation Damage

Three cell lines are used to determine the effectiveness of TPA againstradiation damage: interleukin-3 dependent murine hematopoieticprogenitor cell line, human bone marrow stromal cell line KM101, andbronchial epithelial (IB3) cells. 32D cl 3 interleukin-3 (IL-3)dependent murine hematopoietic progenitor cell line is derived from along-term bone marrow culture of a C3H/HeJ mouse as described inEpperly, 2008. Cells are passaged in 15% WEHI-3 cell conditioned medium(as a source of IL-3), 10% fetal bovine serum (FBS) (HycloneLaboratories, Logan, Utah), and McCoy's supplemented medium. The humanbone marrow stromal cell line KM101 cells are passaged weekly in 24 cm³Falcon plastic flasks in McCoy's 5A modified medium (GIBCO BRL,Gaithersburg, Md.) supplemented with 10% FBS (Hyclone Laboratories,Logan, Utah). IB3 cells are passaged twice weekly in standard Dulbecco'smodified Eagle's medium (DMEM) (Lonza, Allendale, N.J.), supplementedwith 10% FBS (Hyclone laboratories, Logan, Utah), 1% L-glutamine (GIBCOBRL, Gaithersburg, Md.) and 1% penicillin-streptomycin (GIBCO BRL,Gaithersburg, Md.) on uncoated 75 cm³ tissue culture Falcon flasks in a5% CO₂ incubator at 37° C. for 48-72 hours to reach 80% confluency asdescribed in Rwigema, 2011.

Cells from each cell line are suspended at 1×10⁵ cells/mL and irradiatedwith 0 to 8 Gy. TPA is added to the irradiated cells 10 minutes afterirradiation. The cells are then plated in quadruplet and incubated in ahigh-humidity incubator at 37° C. with 95% air: 5% CO2 for 7 days, atwhich time the cells are stained using crystal violet and colonies ofgreater than 50 cells are counted. Each experiment is carried out 3separate times on three separate days. Data are analyzed using linearquadratic and single-hit, multi-target models (See Epperley, 2001). Thedose reduction factor (DRF) for TPA is calculated as the ratio of thedose giving 50% cell survival in the treated group divided by the doseat 50% survival in the control cell group.

Example XIV Protective Effect of TPA Against Damage from Radiation inMice

Adult female C57BL/6 NHsd mice (20 to 22 g, Harlan Sprague Dawley,Chicago, Ill.) (n=15 per group) are irradiated with 9.5 Gy TBI toachieve the (LD 50/30) dose using a Gamma beta irradiation dose rate (74cGy/min) and receive an intraperitoneal injection 10 minutes later of0.125 mg/m² of TPA. The mice are monitored for survival (Rigwema, 2011).

REFERENCES

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We claim:
 1. A method for inducing remission comprising administering toa mammalian subject with cancer, an effective amount of a phorbol esterof Formula I, or a pharmaceutically-acceptable salt, isomer, enantiomer,solvate, hydrate, or polymorph thereof,

wherein R₁ and R₂ may be hydrogen; hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein the lower alkenyl group contains 1 to 7 carbon atoms;

and R₃ is hydrogen or

wherein said effective amount is between about 10 μg to about 1500 μg ofsaid phorbol ester of Formula I is administered at least twice a week,and wherein the phorbol ester of Formula I is administered at least atotal of 15 times, wherein the cancer is selected from the groupconsisting of lymphoma, breast cancer, lung cancer, liver cancer,pancreatic cancer, nasopharyngeal cancer, cervical cancer, acutemyelogenous leukemia, and multiple myeloma.
 2. The method of claim 1,wherein R₁ or R₂ is

the remaining R₁ or R₂ is

and R₃ is hydrogen.
 3. The method of claim 1, wherein the phorbol esteris phorbol 13-butyrate, phorbol 12-decanoate, phorbol 13-decanoate,phorbol 12,13-diacetate, phorbol 13,20-diacetate, phorbol12,13-dibenzoate, phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate,phorbol 12,13-dihexanoate, phorbol 12,13-dipropionate, phorbol12-myristate, phorbol 13-myristate, phorbol 12,13,20-triacetate,12-deoxyphorbol 13-angelate, 12-deoxyphorbol 13-angelate 20-acetate,12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenyl acetate,12-deoxyphorbol 13-phenyl acetate 20-acetate, 12-deoxyphorbol13-tetradecanoate, phorbol 12-tigliate 13-decanoate, 12-deoxyphorbol13-acetate, phorbol 12-acetate, or phorbol 13-acetate.
 4. The method ofclaim 1, wherein the phorbol ester is12-O-tetradecanoylphorbol-13-acetate.
 5. The method of claim 1, whereinthe phorbol ester of Formula I is administered parenterally.
 6. Themethod of claim 1, wherein said phorbol ester of Formula I isadministered at least 30 times.
 7. The method of claim 1, wherein saideffective amount is between about 100 μg to about 300 μg.
 8. The methodof claim 1, further comprising administering at least one secondary oradjunctive therapeutic agent to said subject in a coordinateadministration protocol, simultaneously with, prior to, or after,administration of said phorbol ester to said subject.
 9. The method ofclaim 1, wherein the at least one secondary or adjunctive therapeuticagent is selected from the group consisting of: doxorubicin, vitamin D3,cytarabine, cytosine arabinoside, daunorubicin, cyclophosphamide,gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone,thioguanine, aldesleukin, asparaginase, carboplatin, etoposidephosphate, fludarabine, methotrexate, etoposide, dexamethasone, andcholine magnesium trisalicylate.
 10. The method of claim 9, wherein twosecondary or adjunctive therapeutic agents are administered to saidsubject.
 11. The method of claim 10, wherein the two secondary oradjunctive therapeutic agents are dexamethasone and choline magnesiumtrisalicylate.